Variants of the alpha 1 subunit of human AMPK

ABSTRACT

Variants of the α subunit of AMPK, nucleic acids encoding such variants, and methods for their use are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 10/503,038, filed onJul. 30, 2004 now U.S. Pat. No. 7,208,305, which is a National Stageapplication under 35 U.S.C. §371 and claims benefit under 35 U.S.C.§119(a) of International Application No. PCT/IB03/00807 having anInternational Filing Date of Jan. 31, 2003, which claims the benefit ofpriority of U.S. Provisional Application Ser. No. 60/353,406 having afiling date of Feb. 1, 2002.

TECHNICAL FIELD

The invention relates to protein kinases, and more particularly tovariants of the alpha subunit of the human AMP-activated protein kinase.

BACKGROUND

The AMP-activated protein kinase (AMPK) acts as an intracellularmetabolic sensor in a variety of cells, where it monitors and respondsto variations in the AMP:ATP ratio (Hardie et al., Annu. Rev. Biochem.67:821-855, 1998). Upon activation of AMPK, the enzyme phosphorylates anumber of protein substrates to decrease further ATP usage by the cell.AMPK is a heterotrimeric complex consisting of a catalytic subunit (α)and two associated subunits (β and γ). Both the β and γ subunits arerequired for optimal activity of the α catalytic subunit. The AMPKcomplex is evolutionarily conserved and also can be found in yeast andplants. Mammalian AMPK is composed of different isoforms of subunits:α1, α2, β1, β2, γ1, γ2, and γ3 (Hardie and Hawley, BioEssays23:1112-1119, 2001). Different combinations of isoform subunits areactivated differently in vivo, and most likely also differ in substrateutilization. AMPK activity is found in several tissues, including liver,kidney, muscle, lung, and brain (Cheung et al., Biochem. J. 346:659-669,2000).

AMPK is recognized as a major regulator of lipid biosynthetic pathwaysdue to its role in the phosphorylation and inactivation of key enzymessuch as acetyl-CoA carboxylase (Hardie and Carling, Eur. J. Biochem.246:259-273, 1997). More recent work has suggested that AMPK has a widerrole in metabolic regulation (Winder and Hardie, Am. J. Physiol.277:E1-E10, 1999); this includes fatty acid oxidation, muscle glucoseuptake, expression of cAMP-stimulated gluconeogenic genes such as PEPCKand G6Pase, and expression of glucose-stimulated genes associated withhepatic lipogenesis, including fatty acid synthase, Spot-14, and L-typepyruvate kinase. Chronic activation of AMPK also can induce theexpression of muscle hexokinase and glucose transporters (Glut4),mimicking the effects of extensive exercise training (Holmes et al. J.Appl. Physiol. 87:1990-1995, 1999). The activation of AMPK thus might bea good approach to treat type 2 diabetes; this hypothesis is supportedby the finding that AMPK is the target for metformin, a drug widely usedto treat type 2 diabetes (Zhou et al. J. Clin. Invest. 108:1167-1174,2001).

SUMMARY

The invention is based on the identification of variants of the α1subunit of human AMPK, including splice variants that result ininclusion of an additional exon in the AMPK mRNA and an additional 15amino acid residues in the encoded polypeptide. Such a polypeptide canregulate the activity of the AMPK trimer or alter the substratespecificity of the kinase.

In one aspect, the invention features a purified polypeptide thatincludes a splice variant of the α1 subunit of human AMPK. The purifiedpolypeptide can contain the amino acid sequence of SEQ ID NO:2, or cancontain an amino acid sequence having at least 75% identity to the aminoacid sequence of SEQ ID NO:2. The purified polypeptide can have theamino acid sequence of SEQ ID NO:4.

The invention also features an isolated nucleic acid encoding apolypeptide, wherein the polypeptide is a splice variant of the α1subunit of human AMPK. The isolated nucleic acid can contain anucleotide sequence at least 75% identical to the nucleotide sequence ofSEQ ID NO:1. The isolated nucleic acid sequence can have the nucleotidesequence of SEQ ID NO:3. The invention also features an expressionconstruct containing such nucleic acids.

In another aspect, the invention features a method for identifying anagent capable of modulating the activity of a polypeptide, wherein thepolypeptide is a splice variant of the α1 subunit of human AMPK. Themethod can include contacting a candidate compound with the polypeptideor with a plurality of cells expressing the polypeptide, measuring theeffect of the candidate compound on the activity of the polypeptide, andidentifying the candidate compound as an agent capable of modulating theactivity of the polypeptide if the activity is increased or decreased inthe presence of the compound.

The invention also features a method for identifying an agent capable ofmodulating the activation of a polypeptide, wherein the polypeptide is asplice variant of the α1 subunit of human AMPK. The method can includecontacting a candidate compound with the polypeptide or with a pluralityof cells expressing the polypeptide, measuring the effect of thecandidate compound on the activation of the polypeptide, and identifyingthe candidate compound as an agent capable of modulating the activationof the polypeptide if the activation is increased or decreased in thepresence of the candidate compound.

The invention features a method for identifying an agent capable ofmodulating the amount of a polypeptide produced by a cell, where thepolypeptide is a splice variant of the α1 subunit of human AMPK. Themethod can involve contacting a candidate compound with a plurality ofcells expressing the polypeptide, measuring the effect of the candidatecompound on the amount of the polypeptide produced by the cells, andidentifying the candidate compound as an agent capable of modulating theamount of the polypeptide produced by the cell if the amount isincreased or decreased in the presence of the candidate compound.

In yet another aspect, the invention features an isolated antibodyhaving specific binding affinity for a polypeptide, where thepolypeptide is a splice variant of the α1 subunit of human AMPK.

The invention also features an isolated nucleic acid probe thatspecifically hybridizes to a nucleic acid encoding a splice variant ofthe α1 subunit of human AMPK or the complement of the nucleic acid. Thenucleic acid probe can specifically hybridize to the nucleic acidsequence of SEQ ID NO:1 or a sequence complementary thereto.

The invention also features a method for specifically detecting thepresence of a polypeptide in a sample, where the polypeptide is a splicevariant of the α1 subunit of human AMPK. The method involves contactingthe sample with an antibody described above.

The invention also features a method for specifically detecting thepresence of an AMPK heterotrimer complex in a biological samplecontaining a polypeptide, where the polypeptide is a splice variant ofthe α1 subunit of human AMPK. The method can include contacting thebiological sample with an antibody described above.

The invention also features a method for specifically detecting thepresence of a nucleic acid encoding a splice variant of the α1 subunitof human AMPK. The method can involve contacting a biological samplewith a nucleic acid probe described above.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Other features, objects, andadvantages of the invention will be apparent from the description andfrom the claims.

DETAILED DESCRIPTION

In general, the invention provides polypeptides that are splice variantsof the α1 subunit of AMPK and nucleic acids encoding such polypeptides.As described herein, a polypeptide splice variant of the α1 subunit ofAMPK contains an additional 15 amino acid residues and can result in anAMPK trimer having a different structural surface than an AMPK trimercontaining an α1 subunit that lacks these extra amino acid residues.This may have a direct effect on substrate binding to AMPK. For example,a protein that normally functions as a substrate for AMPK may not retainthe ability to function as a substrate, possibly because newinteractions prevent the substrate from fitting properly into thesubstrate pocket of the kinase. Since AMPK is known to have differentsubstrates that are involved in different signaling and metabolicpathways, the presence of a variant of the α1 subunit could alter theactivity of the kinase in different signaling and metabolic pathways.

A structural model of the core domain of another kinase (Engh andBossemeyer, Advan. Enzyme Regul. 41:124, 2001) indicates that thesubstrate pocket of a kinase consists of two parts, a small substratepocket and a large substrate pocket. The small substrate pockettypically is where the substrate amino acid residue to be phosphorylatedinteracts with the kinase. The large substrate pocket is defined as thebigger part of the cleft between the two subdomains of a kinase coredomain, where the rest of the substrate protein interacts with thekinase during a phosphorylation event or where the substrate proteinwould be in close proximity to the kinase. Without being bound by aparticular mechanism, the polypeptide sequence encoded by the additionalexon identified herein can lie on one side of the larger substratepocket of the kinase cleft. More specifically, this polypeptide sequencecan be found between alpha helix D and alpha helix E (as defined in Enghand Bossemeyer, supra). This could either extend the two existinghelixes or protrude in an enlarged loop linking the two helixes. Thefunction of polypeptide sequence encoded by the additional exon could,in an AMPK trimer complex: (i) alter the substrate specificity of thekinase, and/or (ii) have a regulatory effect on the activity of thekinase.

The presence of a variant of the α1 subunit in an AMPK kinase trimeralso could potentiate the use of a new substrate. The presence of avariant of the α1 subunit could provide the structural requirementsneeded for a protein that is otherwise not a substrate for AMPK to beable to bind to the kinase and thus function as a substrate.

Variants of the α1 subunit of human AMPK could have a role in regulationof the activity of the AMPK trimer. For example, an α1 subunit variantcould affect the ability of AMP to activate the kinase, by directly orindirectly changing the sensitivity for the kinase to AMP. AMPK isactivated by phosphorylation of an upstream AMPK-kinase (AMPKK) and thepresence of a variant of the α1 subunit could affect, by stericconstraints, the ability of AMPKK to phosphorylate AMPK. Furthermore,the presence of a α1 subunit variant could change, also by stericconsiderations, the regulation of AMPK activation by dephosphorylation.

Polypeptides

The present invention provides polypeptides that are variants of the α1subunit of human AMPK. A “polypeptide” refers to a chain of amino acidresidues, regardless of post-translational modification (e.g.,phosphorylation or glycosylation). As used herein, “variants of the α1subunit of human AMPK” have an amino acid sequence that differs from theamino acid sequence of the wild type human AMPK α1 subunit (see GenBankAccession No. NM_(—)006251). Variants can result from, for example, asubstitution, deletion, or insertion within the wild type amino acidsequence.

A variant can be, for example, a naturally occurring splice variant ofthe human AMPK α1 subunit. In one embodiment, a splice variant includesan amino acid sequence encoded by additional exon sequences of the α1subunit of human AMPK. The additional exon sequence can be located, forexample, between exon 3 and exon 4 of the gene encoding the α1 subunitof human AMPK. The additional exon sequence can be the sequence setforth in SEQ ID NO:1.

The present invention further provides variants of the α1 subunit ofhuman AMPK containing the amino acid sequence shown in SEQ ID NO:2 or anamino acid sequence that is at least 75% identical (e.g., at least 80%identical, at least 90% identical, or at least 95% identical), to apolypeptide containing the amino acid sequence of SEQ ID NO:2. Thevariant can have, for example, the amino acid sequence shown in SEQ IDNO:4.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., percentidentity=number of identical positions/total number of positions (e.g.,overlapping positions)×100). In one embodiment, the two sequences arethe same length.

To determine percent sequence identity, a target nucleic acid or aminoacid sequence is compared to the identified nucleic acid or amino acidsequence using the BLAST 2 Sequences (Bl2seq) program from thestand-alone version of BLASTZ containing BLASTN version 2.0.14 andBLASTP version 2.0.14. This stand-alone version of BLASTZ can beobtained from Fish & Richardson's web site (world wide web atfr.com/blast) or the U.S. government's National Center for BiotechnologyInformation web site (world wide web at ncbi.nlm.rih.gov). Instructionsexplaining how to use the Bl2seq program can be found in the readme fileaccompanying BLASTZ.

Bl2seq performs a comparison between two sequences using either theBLASTN or BLASTP algorithm BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. Tocompare two nucleic acid sequences, the options are set as follows: -iis set to a file containing the first nucleic acid sequence to becompared (e.g., C:\seq1.txt); -j is set to a file containing the secondnucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set toblastn; -o is set to any desired file name (e.g., C:\output.txt); -q isset to −1; -r is set to 2; and all other options are left at theirdefault setting. The following command will generate an output filecontaining a comparison between two sequences: C:\Bl2 seq-ic:\seq1.txt-j c:\seq2.txt-p blastn-o c:\output.txt-q−1-r 2. If thetarget sequence shares homology with any portion of the identifiedsequence, then the designated output file will present those regions ofhomology as aligned sequences. If the target sequence does not sharehomology with any portion of the identified sequence, then thedesignated output file will not present aligned sequences.

Once aligned, a length is determined by counting the number ofconsecutive nucleotides from the target sequence presented in alignmentwith sequence from the identified sequence starting with any matchedposition and ending with any other matched position. A matched positionis any position where an identical nucleotide is presented in both thetarget and identified sequence. Gaps presented in the target sequenceare not counted since gaps are not nucleotides. Likewise, gaps presentedin the identified sequence are not counted since target sequencenucleotides are counted, not nucleotides from the identified sequence.

The percent identity over a particular length is determined by countingthe number of matched positions over that length and dividing thatnumber by the length followed by multiplying the resulting value by 100.For example, if (1) a 50 nucleotide target sequence is compared to thesequence set forth in SEQ ID NO:1, (2) the Bl2seq program presents 45nucleotides from the target sequence aligned with a region of thesequence set forth in SEQ ID NO:1 where the first and last nucleotidesof that 45 nucleotide region are matches, and (3) the number of matchesover those 45 aligned nucleotides is 40, then the 50 nucleotide targetsequence contains a length of 45 and a percent identity over that lengthof 89 (i.e., 40/45×100=89).

It will be appreciated that different regions within a single nucleicacid target sequence that aligns with an identified sequence can eachhave their own percent identity. It is noted that the percent identityvalue is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13,and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18,and 78.19 are rounded up to 78.2. It also is noted that the length valuewill always be an integer.

Antibodies

The invention also provides antibodies that have specific bindingaffinity for a polypeptide variant of the α1 subunit of human AMPK.“Antibody” or “antibodies” includes intact molecules as well asfragments thereof that are capable of binding to an epitope of an AMPKα1 subunit. The term “epitope” refers to an antigenic determinant on anantigen to which an antibody binds. Epitopes usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains, and typically have specific three-dimensionalstructural characteristics, as well as specific charge characteristics.Epitopes generally have at least five contiguous amino acids. The terms“antibody” and “antibodies” include polyclonal antibodies, monoclonalantibodies, humanized or chimeric antibodies, single chain Fv antibodyfragments, Fab fragments, and F(ab)₂ fragments. Polyclonal antibodiesare heterogeneous populations of antibody molecules that are specificfor a particular antigen, while monoclonal antibodies are homogeneouspopulations of antibodies to a particular epitope contained within anantigen. Monoclonal antibodies are particularly useful.

In general, an AMPK α1 polypeptide is produced, for example, by chemicalsynthesis or by purification of the native protein and then used toimmunize animals. Various host animals including, for example, rabbits,chickens, mice, guinea pigs, and rats, can be immunized by injection ofthe protein of interest. Depending on the host species, adjuvants can beused to increase the immunological response and include Freund'sadjuvant (complete and/or incomplete), mineral gels such as aluminumhydroxide, surface-active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,and dinitrophenol. Polyclonal antibodies are contained in the sera ofthe immunized animals. Monoclonal antibodies can be prepared usingstandard hybridoma technology. In particular, monoclonal antibodies canbe obtained by any technique that provides for the production ofantibody molecules by continuous cell lines in culture as described, forexample, by Kohler et al. (1975) Nature 256:495-497, the human B-cellhybridoma technique of Kosbor et al. (1983) Immunology Today 4:72, andCote et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030, and theEBV-hybridoma technique of Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc. pp. 77-96 (1983). Such antibodies can be ofany immunoglobulin class including IgM, IgG, IgE, IgA, IgD, and anysubclass thereof. The hybridoma producing the monoclonal antibodies ofthe invention can be cultivated in vitro or in vivo.

A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a mouse monoclonal antibody and a humanimmunoglobulin constant region. Chimeric antibodies can be producedthrough standard techniques.

Antibody fragments that have specific binding affinity for an AMPK α1subunit polypeptide can be generated by known techniques. Such antibodyfragments include, but are not limited to, F(ab′)₂ fragments that can beproduced by pepsin digestion of an antibody molecule, and Fab fragmentsthat can be generated by deducing the disulfide bridges of F(ab′)₂fragments. Alternatively, Fab expression libraries can be constructed.See, for example, Huse et al. (1989) Science 246:1275-1281. Single chainFv antibody fragments are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge (e.g., 15 to 18amino acids), resulting in a single chain polypeptide. Single chain Fvantibody fragments can be produced through standard techniques, such asthose disclosed in U.S. Pat. No. 4,946,778.

Once produced, antibodies or fragments thereof can be tested forrecognition of an AMPK α1 subunit by standard immunoassay methodsincluding, for example, enzyme-linked immunosorbent assay (ELISA) orradioimmuno assay (RIA). See Short Protocols in Molecular Biology. eds.Ausubel et al., Green Publishing Associates and John Wiley & Sons(1992). Suitable antibodies typically have equal binding affinities forrecombinant and native proteins.

Antibodies that have specific binding affinity for an AMPK α1 subunitvariant can be used to detect variants of the α1 subunit of human AMPKin a biological sample. As used herein, a biological sample containscells or cellular material, and can include, for example, urine, blood,cerebrospinal fluid, pleural fluid, sputum, peritoneal fluid, bladderwashings, secretions, oral washings, tissue samples, touch preps, orfine-needle aspirates. Methods for detecting α1 subunit variants includecontacting such a sample with an antibody of the invention. The variantof the α1 subunit can be present in an AMPK heterotrimer complex.

Nucleic Acids

The present invention further provides isolated nucleic acid moleculesencoding variants of the α1 subunit of human AMPK. Nucleic acids of theinvention can contain nucleotide sequences that are at least 75%identical (e.g., at least 80% identical, at least 90% identical, or atleast 95% identical), to the nucleotide sequence of SEQ ID NO:1. Thenucleotide sequence of an isolated nucleic acid sequence may be, forexample, the nucleotide sequence shown in SEQ ID NO:3.

The invention also provides vectors containing nucleic acid sequencesencoding variants of the α1 subunit of human AMPK. As used herein, a“vector” is a replicon, such as a plasmid, phage, or cosmid, into whichanother DNA segment can be inserted so as to bring about the replicationof the inserted segment. The vectors of the invention can be expressionvectors. An “expression vector” is a vector that includes one or moreexpression control sequences, and an “expression control sequence” is aDNA sequence that controls and regulates the transcription and/ortranslation of another DNA sequence.

An expression vector containing a nucleic acid that encodes a variant ofthe human AMPK α1 subunit can be introduced into host cells by any of anumber of techniques including, for example, calcium phosphatetransformation, DEAE-dextran transformation, cationic lipid mediatedlipofection, electroporation, or infection.

The α1 subunit can be expressed in a variety of hosts such as bacteria,plant cells, insect cells, fungal cells, and human and animal cells.Eukaryotic recombinant host cells are particularly useful. Nonlimitingexamples include yeast, mammalian cells including cell lines of human,bovine, porcine, monkey, and rodent origin, and insect cells includingDrosophila and silkworm derived cell lines. Examples of cell linesderived from mammalian species that are commercially available include:L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), HEK 293(ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92),NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616),BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).

The invention further provides nucleic acid probes that can specificallyhybridize to a nucleic acid encoding a variant of the α1 subunit ofhuman AMPK or to the complement of the nucleic acid. Nucleic acid probesthat specifically hybridize to the nucleotide sequences of SEQ ID NO:1and SEQ ID NO:3, or sequences complementary thereto are particularlyuseful. Such nucleic acid probes are useful, for example, for amplifyingand/or detecting a nucleic acid encoding a polypeptide of the invention.

“Specific hybridization” of a nucleic acid probe refers to a nucleicacid that hybridizes only to nucleic acids encoding variants of the α1subunit of human AMPK, or the complements thereof, without hybridizingto nucleic acids encoding related polypeptides. Such hybridizationtypically is carried out under stringent hybridization conditions. Theterm “stringent” when used in conjunction with hybridization conditionsis as defined in the art, i.e., 15-20° C. under the melting point Tm.Preferably the conditions are “highly stringent,” i.e., 5-10° C. underthe melting point Tm. High stringency conditions can include the use oflow ionic strength buffer and a high temperature for washing, forexample, 0.015 M NaCl/0.0015 M sodium citrate (0.1×SSC), 0.1% sodiumdodecyl sulfate (SDS) at 65° C. Alternatively, denaturing agents such asformamide can be employed during hybridization, e.g., 50% formamide with0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mMsodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrateat 42° C. Defining appropriate hybridization conditions is within theskill of the art. See, e.g., Molecular Cloning: A Laboratory Manual, 3rded., Sambrook et al. eds., Cold Spring Harbor Laboratory Press, 2001;DNA Cloning: A practical Approach. Glover & Hames eds., OxfordUniversity Press, 1996; and Nucleic Acid Hybridization: Essentialtechniques, Ross ed. Wiley, 1998.

Methods

The invention provides methods for identifying a therapeutic agentcapable of modulating the activity of a polypeptide, where thepolypeptide is a variant of the α1 subunit of human AMPK. Such methodscall include: (i) contacting a candidate compound with such apolypeptide or a plurality of cells expressing such a polypeptide; and(ii) measuring the effect of the candidate compound on the activity ofthe polypeptide. Modulating the activity of a variant of the α1 subunitof human AMPK can be achieved by, for example, modulating the level ofphosphorylation, substrate specificity, AMP activation, substrateaffinity, or resistance to protein phosphorylases. Modulating theactivity of a variant of the α1 subunit of human AMPK can occur byeither stimulation or inhibition. Agents that can stimulate the activityof a variant of the α1 subunit of human AMPK are particularly useful.

Methods of the invention also can be used to identify a therapeuticagent capable of modulating the activation of a variant of the α1subunit of human AMPK according to the invention. AMPK can beallosterically activated by AMP, or activated by phosphorylation (e.g.,in the activation loop of the α1 subunit). Thus, modulation of theactivation of a variant of the α1 subunit of human AMPK can be achievedthrough, for example, modulating the level of phosphorylation, AMPactivation, or resistance to protein phosphorylases. Methods foridentifying such therapeutic agents can include: (i) contacting acandidate compound with a variant of the α1 subunit of human AMPK or aplurality of cells expressing a variant of the α1 subunit of human AMPK;and (ii) measuring the effect of the candidate compound on theactivation of the variant of the α1 subunit of human AMPK. Modulation ofthe activation of a variant of the α1 subunit of human AMPK can eitherincrease or decrease the activation. Agents capable of increasing theactivation of a variant of the α1 subunit of human AMPK are particularlyuseful.

Assays used to determine the effect of a compound to be tested on theactivity or activation of a variant of the α1 subunit of human AMPK canbe based on measurement of the in vitro phosphorylation by AMPK ofsynthetic peptide substrates as described, for example, by Davies et al.(Eur. J. Biochem. 186:123-128, 1989) and Michell et al. (J. Biol. Chem.271:28445-28450, 1996).

Therapeutic agents that can modulate the amount of a variant of the α1subunit of human AMPK produced by a cell also can be identified usingmethods of the invention. For example, a candidate compound can becontacted with a plurality of cells expressing a variant of the α1subunit of human AMPK and the effect of the candidate compound on theamount of the variant of the α1 subunit of human AMPK that is producedby the plurality of cells can be measured. Modulation of the amount of avariant of the α1 subunit of human AMPK that is produced includesincreasing or decreasing the amount of the polypeptide that is produced.Therapeutic agents capable of increasing the amount of a variant of theα1 subunit of human AMPK that is produced are particularly useful.

The amount of α1 subunit that is produced in cells can be altered, forexample, by modulating transcription, splicing, or translation ofnucleic acids encoding the variant of the α1 subunit of human AMPK.Assays used to determine the effect of a test compound on the amount ofa variant of the α1 subunit of human AMPK can be based on; (i)measurement of the amount of mRNA formed using, e.g., Northern blotanalysis or quantitative real time PCR, (ii) measurement of the amountof protein formed using, e.g., Western blot analysis, or immunochemicalanalysis such as ELISA, or (iii) measurement of activity as describedabove, in cells expressing a variant of the α1 subunit of human AMPK.

Compounds identified by the methods described herein can be used toregulate metabolism. “Regulation of metabolism” as used herein refers tothe ability of the therapeutic agent to mediate cell processes relatedto insulin resistance syndrome and other related disorders, such asnon-insulin dependent diabetes mellitus, dyslipidemia, obesity andatherosclerosis.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Materials and Methods

Synthetic oligonucleotides were designed for use as primers to amplifyand thus clone the coding region of the α1 catalytic subunit of humanAMPK (GenBank accession number NM_(—)006251) by polymerase chainreaction (PCR). The sense primer (AM2sS) with the nucleotide sequence5′-GAGCATGCAGATGGCGACAGCCGAGAA-3′ (SEQ ID NO:5; start codon underlined)contained a 5′ SphI restriction enzyme site and two extra bases on eachend, while the anti-sense primer (AM2as) had the nucleotide sequence5′-TTATTGTGCAAGAATTTTAATTAGAT-3′ (SEQ ID NO:6). First strand cDNA wassynthesized from human skeletal muscle mRNA (M. quadriceps, iliopsoas,and pectoralis major from male and female; Clontech, Palo Alto, Calif.)using the SuperScript II system (Gibco/Invitrogen, Carlsbad, Calif.).PCR was performed using Advantage 2 polymerase (Clontech), a GenAMP PCRSystem 9600 instrument (Perkin Elmer, Wellesley, Mass.), and thefollowing cycles: an initial denaturation at 95° C. for one minute; fivecycles of 94° C. for 15 seconds, 60° C. for 15 seconds, and 72° C. for 2minutes; 38 cycles of 94° C. for 15 seconds, 54° C. for 10 seconds, and72° C. for 2 minutes; and one final segment of 72° C. for 3 minutes. APCR product approximately 1.7 kb in length was purified from agarosegel. The ends of the PCR product were polished using a T4 DNA polymerase(Amersham Pharmacia Biotech, Piscataway, N.J.) for five minutes at roomtemperature and thereafter purified using the QIAquick PCR PurificationKit (Qiagen, Valencia, Calif.). The PCR product was digested with SphI(New England BioLabs, Beverly, Mass.) for 90 minutes, after which thecDNA fragment was once again purified. The digested PCR fragment wasligated into the SphI and SmaI sites of the bacterial expression vectorpQE-32 (Qiagen) and subsequently transformed into electrocompetent DH10bbacteria (Gibco/Invitrogen). Overnight cultures were started fromindividual colonies and mini plasmid preparation was performed using aQIAprep Spin miniprep Kit (Qiagen). DNA sequencing was performed on aMegaBACE 1000 DNA Analysis System (Amersham Biosciences) and DNAsequence analysis was performed using EditView (Applied Biosystems,Foster City, Calif.) and MacVector (Accelrys, Burlington, Mass.)software programs.

Example 2 Identification and Cloning of a Variant of the α1 Subunit ofHuman AMPK

One of the five clones (SEQ ID NO:3) that were isolated as described inExample 1 contained an internal extra nucleotide sequence of 45 bp, ascompared to the published cDNA sequence for the human AMPK α1 subunit(GenBank #NM_(—)006251). These extra 45 bp did not alter the existingreading frame of the α1 subunit of AMPK.

The extra 45 bp correspond to a previously undescribed exon of the humanAMPK α1 subunit, when compared to genome clone #AC008810. This new exonadheres to the AG-exon-GT consensus for exon/intron boundaries(International Human Genome Sequencing Consortium, Nature 409:860-921,2001). The corresponding positions in the rat and mouse genes for theAMPK α1 subunit show significant sequence similarity to the human gene,but contain nucleotide substitutions, deletions, and insertions, as wellas a lack of consensus motifs at exon/intron boundaries. Thesedifferences likely render the corresponding rat and mouse sequencesincapable of forming any exon. The rat sequence was found in GenBank#AC094562, and the mouse sequence was obtained by PCR using mousegenomic DNA and AMPK α1 subunit specific primers.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An isolated nucleic acid encoding a purified polypeptide comprising asplice variant of the αl subunit of human AMP-activated protein kinase(AMPK) having at least 95% identity to the amino acid sequence of SEQ IDNO:4 and comprising an amino acid sequence having at least 93% identityto the amino acid sequence of SEQ ID NO:2, wherein said variantpolypeptide has kinase activity.
 2. The isolated nucleic acid of claim1, said nucleic acid comprising a nucleotide sequence at least 95%identical to the nucleotide sequence of SEQ ID NO:1.
 3. The isolatednucleic acid of claim 1, wherein said nucleic acid has the nucleotidesequence of SEQ ID NO:3.
 4. An expression construct comprising theisolated nucleic acid of claim
 1. 5. The isolated nucleic acid of claim1, wherein said nucleic acid comprises the nucleotide sequence of SEQ IDNO:1.